Higher Percentage Of Blastocysts Research Articles

Ин витро култивиране на говежди ембриони

Embryo cultivation is the longest step during the in vitro embryo production process. In this study, the effect of adding 5% FCS or 3 mg/ml BSA to SOF and KSOM in vitro culture media was investigated. When adding FCS in SOF medium, a better effect on separation was observed (P < 0.05), and the percentage of blastocysts was higher - 58.6%. When BSA was added to SOF medium, the percentage of blastocysts was 40.1% - significantly lower. When cultured in KSOM medium with the addition of FCS, the division rate was lower than when cultured in SOF+FCS and the proportion of blastocysts was also slightly lower. When BSA was added to the KSOM medium, the percentage of blastocysts was almost as in the SOF+BSA group. Аt the conclusion of the conducted experiment, the addition of FCS in KSOM and SOF media is beneficial for in vitro cultivation, resulting in a higher percentage of blastocysts.

Antioxidant effect of bioactive compounds isolated from Syzygium aromaticum essential oil on the in vitro developmental potential of bovine oocytes

Syzygium aromaticum essential oil (EOSA) is an interesting antioxidant supplement for improving bovine in vitro embryo production (IVEP). Therefore, identifying the bioactives in EOSA is essential for increasing embryonic development. In this study, we compared the effect of EOSA to that of its bioactive compounds on nuclear and cytoplasmic maturation, bioenergetic/oxidative stress parameters, and preimplantation embryonic development of bovine oocytes. Viable oocytes (n = 2,272) were matured under six conditions: control (without antioxidant), cysteamine (100 μM CYS), EOSA20 (20 μg/mL EOSA), eugenol (83 μM EUG), β-caryophyllene (19 μM β-CA), and acetyl eugenol (12 μM ACT). In the first experiment, oocytes were evaluated to determine their in vitro maturation (IVM) according to the expansion and viability of cumulus cell layer, presence of the first polar body (1 PB), metaphase II (MII), and mitochondrial distribution. In the second experiment, oocytes were evaluated to determine their bioenergetic/oxidative status based on the levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (ΔΨm). In the third experiment, mature oocytes were parthenogenetically activated (PA), and the embryos were cultured. In the first experiment, no difference was observed in the percentage of MII among groups containing antioxidants (P > 0.05). However, EOSA20, EUG, and CYS improved the percentage of 1 PB oocytes (P < 0.05). Moreover, EUG improved the viability and expansion of cumulus cell layers (P < 0.05). Additionally, cytoplasmic maturation was higher in the EUG and ACT, and mitochondrial aggregation patterns were higher in the EUG, CYS, and ACT than in the other groups (P < 0.05). In the second experiment, ROS levels were reduced in oocytes matured with EUG and CYS (P < 0.05) and GSH levels increased in the same groups (P < 0.05). Oocytes treated with EOSA20, EUG, ACT, and CYS had lower ΔΨm than control. In the third experiment, EUG, EOSA20, and CYS resulted in higher cleavage rates, and EUG and β-CA resulted in more structures with ≥ 8 cells than the other components (P < 0.05). EUG caused a higher percentage of blastocysts than the other components (P < 0.05). EUG, EOSA20, and CYS improved embryo quality (P < 0.05). In conclusion, EUG is the EOSA bioactive compound with the greatest potential for application as an antioxidant and can be commercially applied for IVM of bovine oocytes to result in higher reproducibility and efficiency for IVEP.

P–160 Sibling oocytes cultured in a time-lapse versus benchtop incubator: limited exposure of embryos outside the incubator improves outcomes

Abstract Study question Does the limited exposure of embryos outside the incubator, during evaluation and changeover, have an impact on the blastocyst development, blastocyst quality and euploid outcomes? Summary answer Exposure of embryos outside the incubator, negatively impacts the number, quality and euploidy rate of day 5 blastocysts. What is known already The laboratory environment with its culture conditions is one of the crucial elements of the delicate equation to a successful ART outcome. It has been shown that increased fluctuations in the culture conditions have a considerable impact on the number of blastocysts obtained and cycle outcomes. Compared to conventional benchtop incubators, Time Lapse Technology (TLT) incubators capture images of the embryo and allow morphologic and morphokinetic assessment without disturbance during incubation. Several studies have been published comparing the efficiency, safety and outcome performance between conventional and TLT incubators, however, none of them explored the euploid outcomes. Study design, size, duration An observational sibling oocyte study was performed at ART Fertility Clinics, Abu Dhabi between March 2018 and April 2020 and included data of 796 mature oocytes injected from 42 stimulation cycles. Sibling oocytes were randomly split between 2 different incubators: 12 oocytes were assigned to the twelve wells of the EmbryoscopeTM (ES) and the remaining oocytes were cultured in a conventional benchtop incubator, G185 K-System (KS). Participants/materials, setting, methods Embryos from patients with primary or secondary infertility, who underwent ovarian stimulation for ICSI and PGT-A through NGS on trophectoderm biopsies, were eligible. All patients had at least 16 fresh mature oocytes, randomly allocated to two different incubators after ICSI: 503 (63.2%) oocytes were cultured in ES and 293 (36.8%) in KS. The fertilization, cleavage, useable blastocyst and euploid rates, as well as embryo/blastocyst qualities were assessed to evaluate each incubator’s performance. Main results and the role of chance The fertilization and cleavage rates were similar between incubators. Total useable blastocyst rate (64.8% vs 49.6%, p &amp;lt; 0.001) was significantly higher for embryos cultured in ES, mainly due a higher percentage of blastocysts biopsied on day 5 in ES (67.8% vs 57.0%, p = 0.037), with improved quality (p = 0.008). There was no difference in the total euploid rate between ES and KS (59.9% vs 50.4%, p = 0.314), but a significantly higher euploid rate was seen for blastocysts cultured in ES and biopsied on day 5 (63.5% vs 37.4%, p = 0.001). Day 3 embryo quality and total biopsied blastocyst quality was not different between incubators. No difference was observed in the total useable blastocyst development from good (p = 0.0832) and poor (p = 0.112) quality day 3 cleavage stage embryos. However, when stratifying according to the day of blastocyst development, poor quality embryos on day 3 showed superior blastocyst formation on day 5 when cultured in ES (64.1% vs 39.1% for day 5 and 35.9% vs 60.9% for day 6, p = 0.005). Accordingly, blastocyst formation from poor quality embryos on day 3, was shifted to day 6 for embryos cultured in KS. This difference in the day of blastocyst development was not observed for good quality cleavage stage embryos (p = 0.917). Limitations, reasons for caution The current observational study needs confirmation in a prospective trial and should also include the implantation potential of the euploid blastocysts, which was not followed in the current study. A good prognosis population (≥16 mature oocytes) was studied and may not reflect the outcomes in patients with lower oocyte numbers. Wider implications of the findings: This work builds evidence to the solid introduction of the TLT incubators to the clinical routine, as the reduced exposure of embryos outside the incubator – and hence decreased stress - improves the blastocyst development. Trial registration number NA

Effect of the antral follicle count of Bos taurus × Bos indicus dairy cows on in vitro embryo production

This study aimed to evaluate the potential of Girolando (Bos taurus × Bos indicus) cows with high and low antral follicle counts (AFC) for the in vitro production of bovine embryos (IVEP), as well as the pregnancy rates of the recipients of these embryos. Girolando cows (Bos taurus × Bos indicus) were classified as high and low AFC when they had 35-52 (n = 13) and 11-17 follicles (n = 15), respectively. All animals were subjected to repeated follicular aspiration [Ovum pick-up (OPU)] and subsequent IVEP sessions. The synchronization protocol of the recipients was performed on a random day of the estrous cycle (Day 0) with the implantation of progesterone, estradiol benzoate, and prostaglandin. The high AFC group had higher aspirated oocyte/OPU (42.6 ± 5.2 vs. 14.6 ± 1.9; p &lt; 0.01) and cultured oocyte/OPU (38.1 ± 6.6 vs. 12.3 ± 2.8; p &lt; 0.01) averages as well as a higher blastocyst percentage on D7 (23.0 ± 1.0% vs. 18.4 ± 1.5%; p &lt; 0.05) and higher pregnancy rate (42.7 ± 2.7% vs. 39.7 ± 4.6%; p &lt; 0.05) than the low AFC group. Thus, we can conclude that animals with high AFC had better IVEP success rates than animals with low AFC.

L-ergothioneine supplementation during culture improves quality of bovine in vitro–produced embryos

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro–produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%–40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.

186 THE EFFECT OF SHORT VERSUS LONG TERM ELEVATED NONESTERIFIED FATTY ACID CONCENTRATIONS DURING MURINE IN VITRO FOLLICLE GROWTH ON OOCYTE DEVELOPMENTAL COMPETENCE

Metabolic disorders, like obesity and type 2 diabetes are characterised by lipolysis-linked elevated nonesterified fatty acid (NEFA) concentrations. Exposure to high NEFA concentrations during the final phase of bovine in vitro oocyte maturation (24 h) impairs oocyte developmental competence and subsequent embryo quality. However, because elevated NEFA concentrations in vivo are often present for a longer period of time, our recent research focused on a more in vivo-like long-term NEFA exposure (12 d) of whole murine follicles in vitro. The model covers follicular growth from the early secondary to the antral stage in vitro, including final oocyte maturation after an ovulatory stimulus (OS). Results showed an altered follicular growth and physiology (steroid synthesis, gene expression) and a subsequent reduced oocyte developmental competence. However, it remains unclear what specific time frame in follicular development is the most sensitive to such metabolic insult. Therefore, we hypothesised that chronic elevated NEFA concentrations throughout follicle growth affect oocyte developmental competence more severely than short-term NEFA exposure limited to the final phase of oocyte maturation. The aim was to study the effect of elevated NEFA concentrations 1) during the final phase of follicular oocyte maturation (after the OS) and 2) during the whole period of in vitro murine follicle growth until the antral stage, including final oocyte maturation. Early secondary follicles, isolated from the ovaries of 13-day-old B6CBAF1 mice, were cultured in vitro until the antral stage (3 replicates). Follicles were exposed to: BASAL NEFA mix for 12 days (BASAL-BASAL, control); HIGH stearic acid (SA) for 12 days (SA-SA); HIGH NEFA mix for 12 days (NEFA-NEFA); HIGH SA after the OS (BASAL-SA); and HIGH NEFA mix after the OS (BASAL-NEFA). Oocytes were isolated out of antral follicles 20 hours after the OS, routinely fertilized and presumptive zygotes cultured. Cleavage (n cleaved zygotes/n oocytes) and blastocyst (n blastocysts/n oocytes) rates were documented and analysed by means of binary logistic regression. Cleavage rate was reduced for BASAL-BASAL (37%) compared to BASAL-NEFA embryos (52%; P = 0.045). The BASAL-NEFA treatment (43%) presented with a higher blastocyst percentage than BASAL-BASAL (23%; P = 0.004), NEFA-NEFA (26%; P = 0.037) and SA-SA (15%; P = 0.001) treatments. The BASAL-SA (30%) treatment performed better than the SA-SA treatment (P = 0.049). Even though BASAL-BASAL (control) embryo development was surprisingly low, the results indicate that long-term NEFA exposure during follicle growth in vitro affects oocyte developmental competence more severely than an exposure limited to the final phase of maturation after the OS. The results thus emphasise that the maternal micro-environment throughout follicular growth and not only during final oocyte maturation is essential for optimal oocyte quality.

Identification of Single-Nucleotide Polymorphism in the Progesterone Receptor Gene and Its Association With Reproductive Traits in Rabbits

A total of 598 F2 does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1-8 were amplified and sequenced. One SNP was found in the promoter region, 2464G>A, three SNPs in the 5'-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P &lt; 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P &lt; 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

101 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

In the production of cloned pigs, a large number of nuclear transfer (NT) embryos generally need to be transferred into a single surrogate. Thus, attempts to conduct embryo transfer can be frustrating when either a synchronized surrogate is not available, or enough NT embryos are not produced. This problem would be solved if one could cryopreserve the porcine nuclear transfer embryos. Cryopreservation of porcine embryos has been successful only for in vivo-derived embryos. In vitro-derived porcine embryos are sensitive to chilling, and this sensitivity has been attributed to the lipid droplets in the cytoplasm. In previous reports, the viability of cyropreserved embryos was improved by removal of lipid drops from the cytoplasm. Therefore we designed a procedure to cryopreserve cloned blastocysts by a combination of the open pulled straw (OPS) vitrification method with removal of lipid drops from the oocyte. In vitro-matured MII oocytes were enucleated, and centrifuged (10 000 rpm, 5 min) to polarize the lipid droplets. This was followed by removal of the polarized lipid droplets and transfer of a donor fetal-derived fibroblast cell into the perivitelline space by micromanipulation. After electrical activation and fusion, the NT embryos were cultured in PZM-3 medium with 4 mg/mL BSA. Day 5 and Day 6 blastocysts (manipulation day was Day 0) were vitrified by equilibration with 25 mM HEPES-buffered TCM-199 containing 10% ethylene glycol, 10% DMSO, and 20% fetal calf serum for 2 min, followed by exposure to 20% ethylene glycol and 20% DMSO. Embryos were loaded into an OPS straw and immediately plunged into liquid nitrogen. The process from exposure of embryos to vitrification solution to plunging was 25–30 s. Embryos were thawed by immersing the end of the OPS straw in 0.3 M sucrose in which embryos were kept for 5 min, and then in 0.2 M sucrose for 5 min. Some embryos were cultured in PZM-3 for 12 h to determine the percentage and cell number of re-expanded blastocysts. The others were transferred to the uterus of a surrogate gilt within 3 h of thawing. Lipid removal appeared to have no harmful effect on embryo development and cell number of the blastocysts. Interestingly, a higher blastocyst percentage (28.8%, 178/619) was obtained with NT embryos from which the lipid had been removed as compared to normal NT (19.6%, 44/225; P &lt; 0.01). The cell number (31.2 ± 7.7) of re-expanded blastocysts in the delipation group was comparable with normal NT blastocysts (33.6 ± 14.1, P = 0.33). The survival rate of blastocysts after freezing and thawing was enhanced after delipation (delipation group: 66.7%, 14/21; normal NT group: 21.9%, 9/42; P &lt; 0.01). Two hundred and fourteen delipatized NT blastocysts were transferred to four surrogates after freezing and thawing. Three of the surrogates showed a delayed estrus cycle and one is still pregnant as confirmed by ultrasound scanning. We show that the combination of the OPS vitrification method with removal of lipid drops of oocyte cytoplasm might be an efficient way to cryopreserve porcine NT blastocysts. Funding for this project was from the NIH HL51670 and RR018877 and Food for the 21st Century.

The effect of divergent selection for uterine capacity on prenatal survival in rabbits: maternal and embryonic genetic effects.

The aim of this work was to determine whether prenatal survival depends on the genotype of the mother or of the embryo and to identify the critical periods for prenatal mortality in two lines of rabbits divergently selected by high (H) and low (L) uterine capacity. Does from H (n = 124) and L (n = 115) lines were slaughtered at 72 h of gestation. Embryos recovered at 72 h of gestation were transferred to the oviducts of recipient does from the H (n = 23) and L (n = 19) lines. Each recipient does received eight embryos from the H line into one oviduct and eight embryos from the L line into the other. Recipient does were slaughtered on d 28 of gestation. No differences were found between lines in the embryo recovery either in ovulation rate (OR) or in fertilization rate of ova recovered. Recovery rate was higher for the H line (0.80 vs. 0.72, P < 0.01). The number of embryos recovered, fitting ovulation rate as a covariate, was also higher for the H line (9.74 vs. 8.78, P < 0.05). The H line showed a more advanced embryonic stage of development, having a higher percentage of blastocysts (PB) and a lower percentage of compact morulae (PCM) (38% vs. 20%, P < 0.001 for PB, and 51% vs. 64%, P < 0.01 for PCM). The percentage of early morulae was low and similar in both lines. Neither donor nor recipient lines affected embryonic survival from 72 h to 7 d of gestation. Fetal survival was affected by the recipient line (P < 0.05). An interaction between donor and recipient was found. Embryos from the H donor line had a better fetal survival rate than embryos from the L donor line (P < 0.05) in H recipient females. Within L recipient females, embryos from H and L donor lines showed similar fetal survival. Fetal survival was divided into early (from d 7 to 17 of gestation) and late (from d 17 to 28 of gestation). The high recipient line showed a higher early fetal survival than the L recipient line (P < 0.05). The same effect was observed for late fetal survival, but the difference between H and L recipient lines was lower (P < 0.10). Thus, fetal survival depends mainly on the maternal genotype, and the embryo genotype only affects fetal survival when embryo transfer is performed to a favorable maternal environment. Selection for uterine capacity in rabbits leads to modification of early embryonic survival and of early and late fetal survival, but differences are higher for early than for late fetal survival.

A continuous flow, perifusion culture system for 8- to 16-cell bovine embryos derived from in vitro culture

Eight- to 16-cell stage bovine embryos derived from in vitro maturation and in vitro fertilization were obtained after static culture until 80 to 88 h post insemination. The embryos were cultured in a dynamic (continuous flow) perifusion culture system for 24 h and were further co-cultured on a cumulus cell monolayer in a static CO 2 incubator for an additional 80 to 88 h. Based on preliminary experiments, a serum-free bovine embryo culture medium (BECM) supplemented with 25 mM HEPES was used as the perifusion culture medium (PCM), and the flow rate was fixed within the range of 30 to 38 μl/min in subsequent experiments. In Experiment 1, the combined effects of tubing (silicone vs Tygon ™) and/or culture chamber size (0.2 vs 1.5 ml) on prehatching development of the embryos following 24 h perifusion culture were evaluated in an experiment with a 2 × 2 factorial arrangement. More (P < 0.005) embryos developed to the blastocyst stage with Tygon ™ than with silicone tubing, regardless of chamber size. A higher percentage of blastocysts (29%) was obtained following the perifusion culture by the combination of Tygon ™ tubing and the 1.5-ml culture chamber than by any other combination. In addition, a significantly (P < 0.001) lower mean pH of the perifusate was obtained with Tygon ™ (7.57 to 7.58) than with silicone (7.78 to 7.79) tubing, regardless of chamber size. In Experiment 2, embryo development was compared after culture in the perifusion system and in a static culture system using either serum-free medium (control) or serum-free medium plus a cumulus cell monolayer (co-culture). Percentages of morulae (49 vs 57%) and blastocysts (31 vs 33%) obtained from the control system were not significantly (P > 0.4) higher than the percentages obtained from the perifusion system. However, these proportions were significantly (P < 0.01) increased by co-culturing the embryos on a cumulus cell monolayer in the static incubator for the same intervals (71% morulae and 44% in blastocysts). In conclusion, 8- to 16-cell bovine embryos can develop to the blastocyst stage following 24 h perifusion in the dynamic culture system using a serum-free medium. Physical stress due to flow rate within the range of 30 to 38 μl/min in an air atmosphere has little effect on the development of perifused embryos cultured under this system.

Persistence of the developmental block of in vitro fertilized domestic cat embryos to temporal variations in culture conditions.

A series of studies examined the influence and temporal interaction of energy substrate, media complexity, and tissue co-culture on the development of in vitro fertilized cat embryos and the persistence of the morula-to-blastocyst developmental block. In study I, oocytes were fertilized and cultured for 144 hr in a simple culture medium (modified Krebs Ringer bicarbonate; mKrb), containing either glucose or glutamine, or cultured in mKrb w/ glutamine for the initial 72 hr with transfer to mKrb w/ glucose for the final 72 hr. Fertilization rate, percent development to morulae, and cell number per embryo were similar (P > 0.05) between treatments and blastocyst formation was universally low (< 10%). In Study II, oocytes were fertilized and cultured in either mKrb (w/glucose or glutamine) or in a complex medium, Ham's F10 (w/ 10% fetal bovine serum [FBS]). After 72 hr of initial culture, embryos in mKrb were transferred into Ham's F10. Fertilization rate was lower (P < 0.01) in Ham's F10 but embryo development to the morulae stage and cell number per embryo were comparable (P > 0.05) for all treatments. A higher percentage of blastocysts and morulae becoming blastocysts were observed after initial culture in mKrb w/ glutamine than after initial culture in mKrb w/ glucose. In Study III, oocytes were fertilized and cultured initially in mKrb (w/ glutamine), and then switched to either Ham's F10 or cat oviductal cell monolayers (in Ham's F10). Additional embryos were cultured exclusively in Ham's F10 or on cat oviductal cell monolayers. Fertilization rates were lower (P < 0.05) on oviductal cells but cell number per embryo was similar (P > 0.05) in all treatments. Blastocyst formation was lower (P < 0.05) on oviductal cells than in mKrb-Ham's F10 treatment and was < 20% in all treatments. In summary, while in vitro fertilization-derived cat embryos develop to morulae under a variety of culture conditions, the morula-to-blastocyst developmental block was minimally responsive to alterations in energy substrate and medium complexity or fluctuations in their temporal availability. In addition, oviductal cell culture, alone or in combination with other culture variations, was ineffective in overcoming the developmental block.